Method for producing optically active amine compounds by deracemization

Abstract

Disclosed are methods for producing optically active amino acids and amines. According to the methods, α-keto acids are generated as reaction intermediates, and as a result, ω-transaminase-catalyzed kinetic resolution of racemic amino acids or amines as racemic amine compounds enables the production of optically active amine compounds without the need to use expensive α-keto acids as starting materials. Therefore, the optically active amine compounds are produced at greatly reduced costs. In addition, the optically active amine compounds have high enantiomeric excess.

We claim: 
     
       1. A one-pot method for producing an optically pure amino acid by deracemizing a racemic amino acid with two transaminases of opposite enantioselectivity and a co-substrate dedicated for each transaminase, comprising:
 1) providing a racemic amino acid, an amino acceptor for an amino acid transaminase, and an amino donor for a co-transaminase, to the amino acid transaminase and the ω-transaminase whose enantioselectivities are opposite to each other; 
 2) contacting the racemic amino acid, the amino acceptor, and the amino acid transaminase provided in step 1) to convert one amino acid enantiomer of the racemic amino acid to an α-keto acid; and 
 3) contacting the α-keto acid produced in step 2), the amino donor provided in step 1), and the ω-transaminase provided in step 1) to convert the α-keto acid to the amino acid enantiomer whose chirality is opposite to the amino acid enantiomer reacted in step 2), 
 wherein an (R)-amino acid transaminase and a (S)-selective ω-transaminase are used as the amino acid transaminase and the ω-transaminase, respectively, to deracemize the racemic amino acid into optically pure (S)-amino acid, or 
 wherein a (S)-amino acid transaminase and an (R)-selective ω-transaminase are used as the amino acid transaminase and the ω-transaminase, respectively, to deracemize the racemic amino acid into optically pure (R)-amino acid. 
 
     
     
       2. The one-pot method according to  claim 1 , wherein the (R)-amino acid transaminase is provided in step 1), the (R)-amino acid of the racemic amino acid and the amino acceptor are contacted with the R-amino acid transaminase to produce an α-keto acid in step 2), and the α-keto acid is contacted with the (S)-selective ω-transaminase to produce a (S)-amino acid of the α-keto acid in step 3). 
     
     
       3. The one-pot method according to  claim 1 , wherein the (S)-amino acid transaminase is provided in step 1), the (S)-amino acid of the racemic amino acid and the amino acceptor are contacted with the (S)-amino acid transaminase to produce an α-keto acid in step 2), and the a-keto acid is contacted with the (R)-selective ω-transaminase to produce an (R)-amino acid of the a-keto acid in step 3). 
     
     
       4. The one-pot method according to  claim 1 , wherein the (S)-selective ω-transaminase is an enzyme derived from  Ochrobactrum anthropi  and encoded by the nucleic acid sequence set forth in SEQ ID NO: 1 or an enzyme derived from  Paracoccus denitrificans  and encoded by the nucleic acid sequence set forth in SEQ ID NO: 2. 
     
     
       5. The one-pot method according to  claim 1 , wherein the (R)-selective co-transaminase is an enzyme derived from  Aspergillus terreus  and encoded by the nucleic acid sequence set forth in SEQ ID NO: 3 or an enzyme derived from  Arthrobacter  sp. and encoded by the nucleic acid sequence set forth in SEQ ID NO: 4. 
     
     
       6. The one-pot method according to  claim 1 , wherein the racemic amino acid is selected from the group consisting of racemic alanine, racemic serine, racemic homoserine, racemic norvaline, racemic norleucine, racemic leucine, and mixtures thereof. 
     
     
       7. The one-pot method according to  claim 1 , wherein the amino acceptor is selected from the group consisting of a-ketoglutarate, phenylglyoxylate, phenylpyruvate, and mixtures thereof. 
     
     
       8. The one-pot method according to  claim 1 , wherein the amino donor is selected from the group consisting of isopropylamine, methylbenzylamine, benzylamine, and mixtures thereof. 
     
     
       9. The one-pot method according to  claim 1 , wherein the (R)-selective amino acid transaminase is an enzyme derived from  Bacillus sphaericus  and encoded by the nucleic acid sequence set forth in SEQ ID NO: 5. 
     
     
       10. The one-pot method according to  claim 1 , wherein the (R)-selective amino acid transaminase is an enzyme derived from  Escherichia coli  and encoded by the nucleic acid sequence set forth in SEQ ID NO: 6.